Process and apparatus for obtaining amniotic mesenchymal stem cells from amniotic fluid and cells derived therefrom

Abstract

Methods to purify, culture, and select mesenchymal stem cell (MSC) subpopulations with neonatal quality and adult tissue specificity for use in the production of advanced therapeutic pharmaceuticals.

Description

Technical Field

[0001] The present invention relates to a method for purifying, culturing, and selecting a neonatal-quality mesenchymal stem cell (MSC) subpopulation having tissue specificity for use in the manufacture of advanced therapeutic pharmaceuticals.

Background Art

[0002] Amniotic fluid is the fluid that surrounds and protects the fetus during pregnancy. During the last trimester, amniotic fluid is secreted partly by the fetal lungs and partly by the fetal urine. Amniotic fluid is ingested orally and absorbed by the fetal intestine and re-enters the fetal circulation. Term amniotic fluid is composed of water containing electrolytes, but also contains proteins, carbohydrates, lipids, phospholipids, and urea. In addition to metabolic waste products, amniotic fluid also contains fetal cells, as well as other substances that are shed from the skin by friction, such as hair and vernix, a fatty deposit that covers the skin of the newborn at birth. The tissue interfaces in contact with amniotic fluid contribute to the contents of amniotic fluid, which contain cellular material. The lungs are the largest of these surfaces and also secrete pulmonary surfactant into the TAF. The oral and nasal mucosa, eyes, and urinary tract are other such surfaces that have non-keratinized epithelial interfaces in phase contact with amniotic fluid.

[0003] Mesenchymal stem cells (MSCs) are found in almost all tissues and are mainly present in the perivascular niche. As understood by those skilled in the art, mesenchymal stem cells are pluripotent stromal cells that can differentiate into a number of cell types and have anti-inflammatory and angiogenic properties to direct tissue repair processes, and thus mesenchymal stem cells have become valuable for therapeutic treatment. Full-term amniotic fluid (TAF) collected during cesarean section contains many valuable cells, including MSCs. However, the extraction and expansion of MSCs have not been previously carried out on a large scale due to the difficulties associated with the aseptic collection, processing of TAF, as well as the identification and extraction of MSCs. Furthermore, certain subpopulations of MSCs may be particularly suitable for use in the manufacture of therapeutic agents. Previously, to obtain MSCs, MSCs sourced from adult bone marrow, adult adipose tissue, or neonatal birth-related tissues including placenta, umbilical cord, and umbilical cord blood have been widely used. MSCs from these neonatal tissues may have additional capabilities compared to MSCs derived from adult sources. In fact, several studies have reported superior biological properties such as improved proliferative capacity, lifespan, and differentiation ability of MSCs from birth-related tissues compared to adult-derived MSCs. However, none of these neonatal MSC sources have the corresponding tissues or organs in the adult body. Therefore, neonatal-quality MSCs with tissue specificity are highly beneficial. Furthermore, the acquisition of fetal materials may be associated with adverse effects on the infant. For example, in the collection of umbilical cord blood, it has been shown that as much umbilical cord blood as possible needs to be returned to the infant for improved survival rate, growth, and development of fine motor skills. On the other hand, amniotic fluid is currently regarded as medical waste to be discarded. Therefore, both ethical and practical incentives for harvesting such underutilized resources are clear.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

[0005] Certain disclosed examples relate to devices, cells, methods, and systems for obtaining amniotic mesenchymal stem cells from amniotic fluid and cells derived therefrom. It will be understood by those skilled in the art that the application of the devices, methods, and systems described herein is not limited to a particular cell or tissue type. Further examples are described below. In one aspect, the present disclosure is a method for obtaining amniotic mesenchymal stem cells from amniotic fluid, comprising providing term amniotic fluid (TAF), removing particulate matter from the TAF to obtain purified TAF cells, performing adhesion selection on the purified TAF cells to obtain TAF adherent cells, passaging the TAF adherent cells to obtain TAF mesenchymal stem cells (TAF MSC), TBC1 domain family member 3K (TBC1D3K), allograft inflammatory factor 1-like (AIF1L), cadherin-related family member 1 (CDHR1), sodium / potassium-transporting ATPase interacting 4 (NKAIN4), ATP-binding cassette subfamily B member 1 (ABCB1), plasma membrane vesicle-associated protein (PLVAP), mesothelin (MSLN), L1 cell adhesion molecule (L1CAM), hepatitis A virus cellular receptor 1 (HAVCR1), mal, T cell differentiation protein 2 (gene / pseudogene) (MAL2), SLAM family member 7 (SLAMF7), double C2 domain beta (DOC2B), endothelial cell adhesion molecule (ESAM), gamma-aminobutyric acid receptor beta1 subunit type A (GABRB1), cadherin 16 (CDH16), immunoglobulin superfamily member 3 (IGSF3), desmocollin 3 (DSC3), regulator of hemoglobinization and erythrocyte cell proliferation (RHEX), potassium voltage-gated channel interacting protein 1 (KCNIP1), CD70 molecule (CD70), GDNF family receptor alpha1 (GFRA1), Crumbs cell polarity complex component 3 (CRB3), claudin 1 (CLDN1), novel transcript (AC118754.1) Selecting TAF MSCs that express a marker selected from the group consisting of sodium voltage-gated channel alpha subunit 5 (SCN5A), fibroblast growth factor receptor 4 (FGFR4), potassium two pore domain channel subfamily K member 3 (KCNK3), dysferlin (DYSF), ephrin-A1 (EFNA1), potassium inwardly rectifying channel subfamily J member 16 (KCNJ16), membrane-bound RING-CH type finger 1 (MARCHF1), synaptotagmin-like 1 (SYTL1), calsyntenin-2 (CLSTN2), integrin subunit beta-4 (ITGB4), vesicle-associated membrane protein 8 (VAMP8), G protein-coupled receptor class C group 5 member C (GPRC5C), CD24 molecule (CD24), cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2), cadherin 8 (CDH8), glutamate receptor-interacting protein 1 (GRIP1), dematin actin-binding protein (DMTN), F11 receptor (F11R), cell adhesion molecule 1 (CADM1), cadherin 6 (CDH6), coagulation factor II thrombin receptor-like 2 (F2RL2), LY6 / PLAUR domain-containing 1 (LYPD1), solute carrier family 6 member 6 (SLC6A6), desmoglein 2 (DSG2), adhesion G protein-coupled receptor G1 (ADGRG1), cholecystokinin A receptor (CCKAR), oxytocin receptor (OXTR), integrin subunit alpha-3 (ITGA3), adhesion molecule with Ig-like domain 2 (AMIGO2), cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), EPH receptor B-2 (EPHB2).

[0006] In another aspect, the present disclosure provides an isolated cell obtainable by the method according to the present disclosure, the cell being TBC1 domain family member 3K (TBC1D3K), allograft inflammatory factor 1-like (AIF1L), cadherin-related family member 1 (CDHR1), sodium / potassium transporting ATPase interacting 4 (NKAIN4), ATP-binding cassette subfamily B member 1 (ABCB1), plasma membrane vesicle-associated protein (PLVAP), mesothelin (MSLN), L1 cell adhesion molecule (L1CAM), hepatitis A virus cellular receptor 1 (HAVCR1), mal, T cell differentiation protein 2 (gene / pseudogene) (MAL2), SLAM family member 7 (SLAMF7), double C2 domain beta (DOC2B), endothelial cell adhesion molecule (ESAM), gamma-aminobutyric acid receptor beta1 subunit (GABRB1), cadherin 16 (CDH16), immunoglobulin superfamily member 3 (IGSF3), desmocollin 3 (DSC3), regulator of hemoglobinization and erythrocyte cell proliferation (RHEX), potassium voltage-gated channel interacting protein 1 (KCNIP1), CD70 molecule (CD70), GDNF family receptor alpha1 (GFRA1), Crumbs cell polarity complex component 3 (CRB3), claudin 1 (CLDN1), novel transcript (AC118754.1), Sodium voltage-gated channel alpha subunit 5 (SCN5A), fibroblast growth factor receptor 4 (FGFR4), potassium two pore domain channel subfamily K member 3 (KCNK3), dysferlin (DYSF), ephrin-A1 (EFNA1), potassium inwardly rectifying channel subfamily J member 16 (KCNJ16), membrane-bound RING-CH type finger 1 (MARCHF1), synaptotagmin-like 1 (SYTL1), calsyntenin-2 (CLSTN2), integrin subunit beta-4 (ITGB4), vesicle-associated membrane protein 8 (VAMP8), G protein-coupled receptor class C group 5 member C (GPRC5C), CD24 molecule (CD24), cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2), cadherin-8 (CDH8), glutamate receptor-interacting protein 1 (GRIP1), dematin actin-binding protein (DMTN), F11 receptor (F11R), cell adhesion molecule 1 (CADM1), cadherin-6 (CDH6), coagulation factor II thrombin receptor-like 2 (F2RL2), LY6 / PLAUR domain-containing 1 (LYPD1), solute carrier family 6 member 6 (SLC6A6), desmoglein-2 (DSG2), adhesion G protein-coupled receptor G1 (ADGRG1), cholecystokinin A receptor (CCKAR), oxytocin receptor (OXTR), integrin subunit alpha-3 (ITGA3), adhesion molecule with Ig-like domain 2 (AMIGO2), cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), EPH receptor B2 (EPHB2), and express a surface marker selected from the group consisting of...

[0007] In certain examples, a method for obtaining term amniotic fluid-derived mesenchymal stem cells (TAF MSCs) from term amniotic fluid is providing term amniotic fluid (TAF), removing particulate matter from the TAF to obtain purified TAF cells, performing adhesion selection of the purified TAF cells to obtain TAF adherent cells, passaging the TAF adherent cells to obtain a population of cells containing TAF MSCs, Selecting TAF MSCs from a population as cells that express at least one Group A surface marker selected from the group consisting of TBC1 domain family member 3K, allograft inflammatory factor 1-like, cadherin-related family member 1, sodium / potassium transporting ATPase interacting 4, ATP-binding cassette subfamily B member 1, plasma membrane vesicle-associated protein, mesothelin, L1 cell adhesion molecule, hepatitis A virus cellular receptor 1, mal, T cell differentiation protein 2 (gene / pseudogene), SLAM family member 7, double C2 domain beta, endothelial cell adhesion molecule, type A gamma-aminobutyric acid receptor beta1 subunit, cadherin 16, immunoglobulin superfamily member 3, desmocollin 3, regulator of hemoglobinization and erythrocyte cell proliferation, potassium voltage-gated channel interacting protein 1, CD70 molecule, GDNF family receptor alpha1, Crumbs cell polarity complex component 3, claudin 1, novel transcript, sodium voltage-gated channel alpha subunit 5, fibroblast growth factor receptor 4, potassium two pore domain channel subfamily K member 3, dysferlin, ephrin A1, potassium inwardly rectifying channel subfamily J member 16, membrane-bound RING-CH type finger 1, synaptotagmin-like 1, calsyntenin 2, integrin subunit beta4, vesicle-associated membrane protein 8, G protein-coupled receptor class C group 5 member C, CD24 molecule, cadherin EGF LAG seven-pass G-type receptor 2, cadherin 8, glutamate receptor interacting protein 1, dematin actin-binding protein, F11 receptor, cell adhesion molecule 1, cadherin 6, coagulation factor II thrombin receptor-like 2, LY6 / PLAUR domain-containing 1, solute carrier family 6 member 6, desmoglein 2, adhesion G protein-coupled receptor G1, cholecystokinin A receptor, oxytocin receptor, integrin subunit alpha3, adhesion molecule with Ig-like domain 2, cadherin EGF LAG seven-pass G-type receptor 1, and EPH receptor B2, thereby obtaining TAF MSCs.

[0008] In some examples, selecting the TAF MSCs can include selecting TAF MSCs with reduced expression of markers selected from the group consisting of IL13RA2, CLU, TMEM119, CEMIP, LSP1, GPNMB, FAP, CRLF1, MME, CLMP, BGN, DDR2. Removing particulate matter can include filtering the TAFs and centrifuging. Performing adhesion selection of the purified TAF cells can include adhering the purified TAF cells to a surface coated with a vitronectin-based substrate. The selection step can be performed using fluorescence-activated cell sorting (FACS). The selection step can be performed using an antibody against either a marker or a surface marker. The selection step can include selecting TAF MSCs that express at least two markers from group A surface markers. The selection step can include selecting TAF MSCs that express at least three markers from group A surface markers. The selection step can include selecting TAF MSCs that express at least four markers from group A surface markers. The selection step may include a plurality of sorting steps, each sorting step including assigning the TAF MSCs to a first output group or a second output group according to the set of markers expressed or not expressed by each TAF MSC.

[0009] In some examples, the selection step can include a first sorting step for assigning TAF MSCs that express group A surface markers to a first output group, and a second sorting step for assigning TAF MSCs from the first output group that express a second set of markers to a second output group.

[0010] In certain examples, a method for obtaining term amniotic fluid-derived term amniotic fluid lung mesenchymal stem cells (lung TAF MSCs) is providing term amniotic fluid (TAF), removing particulate matter from the TAF to obtain purified TAF cells, performing adhesion selection of the purified TAF cells to obtain TAF adherent cells, The TAF then subcultures the cells to obtain a population of cells containing lung TAF MSCs, selects the TAF lung MSCs from the population as cells that express at least one group B surface marker selected from the group consisting of PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3, SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1, CD248, DDR2, PCDH18, LRRC38, and CRLF1, thereby obtaining TAF lung mesenchymal stem cells, which may be included.

[0011] The selection of the lung TAF MSCs may include excluding MSCs that express markers selected from the group consisting of CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR. The selection step may include selecting TAF MSCs that express at least two surface markers from the group B surface markers. The selection step may include selecting TAF MSCs that express at least three surface markers from the group B surface markers. The selection step may include selecting TAF MSCs that express at least four surface markers from the group B surface markers. The selection step may include selecting TAF MSCs that express surface markers selected from the group consisting of CD248, DDR1, and LRRC38. The selection step may include selecting TAF MSCs that express CD248. The selection step may include selecting TAF MSCs that express CD248 in combination with markers selected from the group consisting of DDR1 and LRRC38. The selection step may include selecting TAF MSCs that express CD248, DDR1, and LRRC38. In some examples, isolated term amniotic fluid (TAF) mesenchymal stem cells can be obtained by the above method, and the cells express at least one group A surface marker.

[0012] In some instances, an isolated population of term amniotic fluid (TAF) mesenchymal stem cells can express at least one Group A surface marker selected from the group consisting of TBC1 domain family member 3K, allograft inflammatory factor 1-like, cadherin-related family member 1, sodium / potassium transporting ATPase interacting 4, ATP-binding cassette subfamily B member 1, plasma membrane vesicle-associated protein, mesothelin, L1 cell adhesion molecule, hepatitis A virus cellular receptor 1, mal, T cell differentiation protein 2 (gene / pseudogene), SLAM family member 7, double C2 domain beta, endothelial cell adhesion molecule, type A gamma-aminobutyric acid receptor beta1 subunit, cadherin 16, immunoglobulin superfamily member 3, desmocollin 3, regulator of hemoglobinization and erythrocyte cell proliferation, potassium voltage-gated channel interacting protein 1, CD70 molecule, GDNF family receptor alpha1, Crumbs cell polarity complex component 3, claudin 1, novel transcript, sodium voltage-gated channel alpha subunit 5, fibroblast growth factor receptor 4, potassium two pore domain channel subfamily K member 3, dysferlin, ephrin A1, potassium inwardly rectifying channel subfamily J member 16, membrane-bound RING-CH type finger 1, synaptotagmin-like 1, catenin beta2, integrin subunit beta4, vesicle-associated membrane protein 8, G protein-coupled receptor class C group 5 member C, CD24 molecule, cadherin EGF LAG seven-pass G-type receptor 2, cadherin 8, glutamate receptor interacting protein 1, dematin actin-binding protein, F11 receptor, cell adhesion molecule 1, cadherin 6, coagulation factor II thrombin receptor-like 2, LY6 / PLAUR domain-containing 1, solute carrier family 6 member 6, desmoglein 2, adhesion G protein-coupled receptor G1, cholecystokinin A receptor, oxytocin receptor, integrin subunit alpha3, adhesion molecule with Ig-like domain 2, cadherin EGF LAG seven-pass G-type receptor 1, and EPH receptor B2.

[0013] In some examples, the composition can include an isolated population of the above term-term amniotic fluid (TAF) mesenchymal stem cells and a pharmaceutically acceptable carrier for TAF MSCs. The isolated term-term amniotic fluid (TAF) mesenchymal lung stem cells obtainable by the above method can express at least one group B surface marker selected from the group consisting of PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3, SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1, CD248, DDR2, PCDH18, and CRLF1. In certain examples, the isolated population of term-term amniotic fluid (TAF) lung mesenchymal stem cells can express at least one group B surface marker.

[0014] In some examples, a method for obtaining term-term amniotic fluid kidney mesenchymal stem (kidney TAF MSC) cells from term-term amniotic fluid is providing term-term amniotic fluid (TAF); removing particulate matter from the TAF to obtain purified TAF cells; performing adhesion selection on the purified TAF cells to obtain TAF adherent cells; passaging the TAF adherent cells to obtain a population of cells containing TAF kidney MSCs; selecting TAF kidney MSCs from the population as cells expressing at least one group C surface marker selected from the group consisting of HAVCR1, CD24, CLDN6, ABCB1, SHISA9, CRB3, AC118754.1, ITGB6, CDH1, LSR, EPCAM, AJAP1, ANO9, CLDN7, EFNA1, MAL2, F11R, L1CAM, GFRA1, IGSF3, TNF, MMP7, FOLR1, TGFA, C3, TNFSF10, PDGFB, and WWC1, thereby obtaining TAF kidney MSCs.

[0015] In certain examples, an isolated population of term amniotic fluid (TAF) kidney mesenchymal stem cells (kidney TAF MSCs) can express at least one Group C surface marker selected from the group consisting of HAVCR1, CD24, CLDN6, ABCB1, SHISA9, CRB3, AC118754.1, ITGB6, CDH1, LSR, EPCAM, AJAP1, ANO9, CLDN7, EFNA1, MAL2, F11R, L1CAM, GFRA1, IGSF3, TNF, MMP7, FOLR1, TGFA, C3, TNFSF10, PDGFB, and WWC1.

[0016] The composition can include an isolated population of term amniotic fluid (TAF) kidney mesenchymal stem cells as described above. In some examples, a method for obtaining term amniotic fluid skin mesenchymal stem cells (skin TAF MSCs) from term amniotic fluid is providing term amniotic fluid (TAF), removing particulate matter from the TAF to obtain purified TAF cells, performing adhesion selection of the purified TAF cells to obtain TAF adherent cells, passaging the TAF adherent cells to obtain a population of cells comprising TAF skin MSCs, selecting skin TAF MSCs from the population as cells that express at least one Group D surface marker selected from the group consisting of TNFSF18, PCDH19, NCAM2, TNFSF4, CD248, DDR2, HTR2B, PCDH18, SULF1, MME, ADGRA2, DCSTAMP, PDGFRA, UNC5B, SCUBE3, CEMIP, BDKRB1, FLT1, BDKRB2, FAP, CASP1, and SRPX2, and obtaining the TAF skin MSCs, and can include.

[0017] In certain examples, an isolated population of term amniotic fluid (TAF) skin mesenchymal stem cells (skin MSCs) can express at least one D-group surface marker selected from the group consisting of TNFSF18, PCDH19, NCAM2, TNFSF4, CD248, DDR2, HTR2B, PCDH18, SULF1, MME, ADGRA2, DCSTAMP, PDGFRA, UNC5B, SCUBE3, CEMIP, BDKRB1, FLT1, BDKRB2, FAP, CASP1, and SRPX2. The composition can include the isolated population of term amniotic fluid (TAF) skin mesenchymal stem cells described above and a pharmaceutically acceptable carrier for the TAF skin MSCs.

[0018] In some examples, a method for obtaining term amniotic fluid neural mesenchymal stem cells (neural TAF MSCs) from term amniotic fluid is providing term amniotic fluid (TAF), removing particulate matter from the TAF to obtain purified TAF cells, performing adhesion selection of the purified TAF cells to obtain TAF adherent cells, passaging the TAF adherent cells to obtain a population of cells comprising TAF neural MSCs, selecting TAF neural MSCs from the population as cells that express at least one E-group surface marker selected from the group consisting of HAVCR1, ACKR3, OSCAR, C3, SIRPB1, SLC6A6, CCKAR, TNFSF10, CLSTN2, TENM2, SFRP1, PIK3IP1, SCNN1D, CLDN11, ALDH3B1, and ITGB4, thereby obtaining TAF neural MSCs.

[0019] In some examples, an isolated population of term amniotic fluid (TAF) neural mesenchymal stem cells (neural TAF MSCs) can express at least one group E surface marker selected from the group consisting of HAVCR1, ACKR3, OSCAR, C3, SIRPB1, SLC6A6, CCKAR, TNFSF10, CLSTN2, TENM2, SFRP1, PIK3IP1, SCNN1D, CLDN11, ALDH3B1, and ITGB4. The composition can include the isolated population of term amniotic fluid (TAF) neural mesenchymal stem cells described above and a pharmaceutically acceptable carrier for the TA neural MSCs. In certain aspects, the present disclosure provides methods and apparatuses for isolating term amniotic fluid (TAF) mesenchymal stem cells and compositions comprising TAF mesenchymal stem cells that include one or more of the features described above and / or in the figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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Modes for Carrying Out the Invention

[0021] Detailed Description of Preferred Embodiments A method for purifying, culturing, and selecting MSC subpopulations with neonatal quality and adult tissue specificity is summarized in Figure 1 and detailed below. The examples disclosed herein relate to devices and methods for harvesting, purifying, isolating, proliferating, differentiating, and maturing amniotic fluid-derived cells. The examples disclosed herein are not limited to the harvesting of a particular type of amniotic fluid-derived cells, and the techniques disclosed herein are widely applicable to different cells and tissues.

[0022] Amniotic fluid collection Amniotic fluid can be collected to generate term amniotic fluid (TAF) according to the method described in U.S. Patent Application No. 14 / 776,499 (corresponding to US2016 / 0030489), the entire contents of which are incorporated herein by reference. Specifically, Figure 2 is a block diagram of an example of a method 300 for amniotic fluid collection according to an exemplary example of the present invention. It should be understood that method 300 may include any number of additional or alternative tasks. The tasks shown in Figure 3 need not be performed in the order shown, and method 300 may be incorporated into a more comprehensive procedure or process having additional features not detailed herein.

[0023] As shown in FIG. 2, method 300 may include, for example, making an incision in the uterine wall 301 of a pregnant mother during cesarean section. Step 301 may be performed by a standard doctor's scalpel. As also shown in FIG. 2, method 300 may include inserting an amniotic fluid sampler 302 through the incision in the uterine wall created in step 301. Method 300 also includes penetrating the amnion 303 using the amniotic fluid sampler of step 302. Step 303 may also include penetrating the chorion. In one aspect, the tip is inserted to a depth of 10 cm. In some examples, the tip is inserted to a depth of about 3 cm to about 30 cm. In some examples, the tip is inserted to a depth of about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, about 11 cm, about 12 cm, about 13 cm, about 14 cm, about 15 cm, about 16 cm, about 17 cm, about 18 cm, about 19 cm, about 20 cm, about 21 cm, about 22 cm, about 23 cm, about 24 cm, about 25 cm, about 26 cm, about 27 cm, about 28 cm, or about 29 cm.

[0024] Method 300 further includes collecting amniotic fluid 304 from the amniotic sac using the amniotic fluid sampler of step 302. Step 304 may include initiating a siphon to transfer the amniotic fluid to the collection chamber of the amniotic fluid sampler, such as by opening the inlet valve of the amniotic fluid sampler. Step 304 may also include positioning the collection chamber of the amniotic fluid sampler below the inlet of the amniotic fluid sampler. Step 304 may also include applying a negative pressure source to the outlet of the amniotic fluid sampler to initiate the transfer of the amniotic fluid. Step 304 may include repositioning the inlet of the amniotic fluid sampler to substantially collect all of the available amniotic fluid.

[0025] Finally, method 300 includes removing the amniotic fluid sampler 905 from the amniotic sac. Step 905 may include closing the inlet valve of the amniotic fluid sampler. In one example, no blood is visible in the collected substance. Step 905 may also include emptying the collection system for further use / processing and sterilizing the exterior of the entire device. In one example, the exterior is sterilized using 70% ethanol, such that sterilization can be maintained, for example, for any post-treatment steps such as isolation of cell material according to the present invention and laminar flow bench setups for fluid storage.

[0026] In one example, the amniotic fluid sampling procedure is performed in less than 1 minute. In one example, the amniotic fluid sampling procedure is performed in 1 - 2 minutes. In one example, the amniotic fluid sampling procedure is performed within 3 minutes. In one example, the method is simplified compared to standard surgical procedures for cesarean section, for example, by preventing leakage of amniotic fluid into the surgical wound and improving visibility and physical access. In one example, the fetal skin is not affected by the device tip.

[0027] Purification Terminated amniotic fluid (TAF) is purified by filtering the terminated amniotic fluid to remove vernix caseosa. The term "terminated amniotic fluid" is used herein and elsewhere in the present disclosure, but it should be understood that the methods, processes, and devices of the present disclosure are applicable to not only terminated amniotic fluid but all amniotic fluids. Terminated amniotic fluid can be, for example, amniotic fluid collected during a terminated cesarean delivery using a system based on a closed catheter. For the purposes of this specification, "terminated amniotic fluid" can be amniotic fluid collected during a scheduled cesarean section after 37 weeks of gestation or a scheduled cesarean section near term, for example, after 36 weeks of gestation. Preferably, terminated amniotic fluid is collected during a scheduled cesarean section after 37 weeks of gestation.

[0028] Figure 3 is a schematic diagram of an apparatus 100 for filtering amniotic fluid according to an example. The amniotic fluid contains fetal or amniotic cells derived from the amniotic sac, such as mesenchymal stem cells. The amniotic fluid also contains other substances that have been shed from the skin by friction, such as hair and vernix caseosa. Substances other than amniotic cells are referred to herein as particulate matter and may include meconium, blood clots, etc. Particulate matter can be considered all those larger than 20 μm. For the purpose of filtration, it may be particularly advantageous to treat all those larger than 30 μm or 50 μm as particulate matter. Optionally, all those larger than the target amniotic cells can be treated as particulate matter. Thus, amniotic fluid generally contains a mixture of amniotic cells and particulate matter. The apparatus 100 includes a filter 101 for filtering particulate matter from the amniotic fluid and a chamber 102 surrounding the filter 101. The chamber 102 includes a fluid inlet 103 and a fluid outlet 104. The chamber 102 surrounding the filter 101 isolates the filter 101 from the environment surrounding the chamber 102, and thus, it should be interpreted that there is no fluid communication between the amniotic fluid in the chamber 102 and the environment. Thus, fluid communication through the chamber 102 is controlled via the fluid inlet 103 and the fluid outlet 104 in the example of FIG. 3. The filter 101 is mounted inside the chamber 102 between the fluid inlet 103 and the fluid outlet 104. FIG. 12 shows an example of a cross-section A-A of the circular chamber 102 and the filter 101 as shown in FIG. 12. However, it should be understood that the chamber 102 and the filter 101 can have various shapes for optimization for different applications. The apparatus 100 includes an inlet connector 105 arranged to form a sealed connection between the fluid inlet 103 and an amniotic fluid sample source 201 (shown in FIG. 4). FIG. 4 shows a schematic example of such an amniotic fluid source 201. By having an inlet connector 105 connected to the fluid inlet 103 and configured to provide a direct sealed connection between the fluid inlet 103 and the amniotic fluid source 201, exposure to contaminants is minimized and efficient aseptic processing of the amniotic fluid is provided. This facilitates obtaining amniotic cells and enables post-filtration processing with improved quality standards. Thus, the manufacturing process of sterile pharmaceuticals is facilitated.For example, the preparation of surfactant molecules can be facilitated. The apparatus 100 provides an improvement in the function of amniotic stem cells, such as an improvement in the engraftment period after transplantation. Such an improved process is made possible by having a filter 101 surrounded by a chamber 102 and an inlet connector 105 arranged to form a sealed connection between the fluid inlet 103 of the chamber 102 and the amniotic fluid sample source 201. The risk of exposing amniotic stem cells to contaminants such as bacteria and viruses is thus reduced. Exposure to oxygen is also minimized, reducing the formation of oxygen free radicals that can adversely affect the function of the stem cells.

[0029] FIG. 3 shows an example where the inlet connector 105 includes a tube 105 connected to the fluid inlet 103 at the first sealed connection portion 114. The inlet connector 105 can form a sealed connection with the fluid inlet 103 using a press-fit connection, an adhesive, a clamp, or other fixing elements. In another example, as schematically shown in FIG. 4, the inlet connector 105 is a continuous extension of the fluid inlet 103 without separate fixing elements, for example, by being formed as a single part by molding or other material forming techniques. FIGS. 3 and 4 show a second connector 115 configured to form a sealed connection with a sample source 201 such as a container or bag 201 containing amniotic fluid. The second connector 115 can comprise a releasable press-fit connection, a clamp, or a combination thereof, or other releasable fixing elements. The chamber 102, the filter 101, the fluid inlet 103, the fluid outlet 104, and the inlet connector 105 can be provided as a kit within a sterile package, for example, as a disposable kit. Such a kit, i.e., the device 100, provides an easy and improved process for obtaining amniotic stem cells in this way. Thus, in use, the amniotic fluid passes through the filter 101 when flowing from the fluid inlet 103 to the fluid outlet 104. As a result, particulate matter is deposited on the filter 101, and the amniotic fluid containing amniotic cells flows through the fluid outlet 104. As seen in the example of FIG. 12, the filter 101 can be connected to the inner wall 113 of the chamber 102 around its peripheral edge 116. This avoids the amniotic fluid passing from the inlet 103 to the outlet 104 without being filtered. The filter 101 can be tensioned or otherwise supported so that bending or curving of the filter 101 within the chamber 102 is avoided. This maintains the mesh diameter or pore system defined across the entire area of the filter 101, and thus maintains the defined filtration characteristics. Maintaining the defined mesh diameter or pore system also reduces the risk of clogging the filter 101. As a result, long-term performance can be improved.

[0030] The device 100 may comprise an outlet connector 106 so as to form a sealed connection between the outlet and an amniotic cell receiving device 202 such as a centrifuge or other amniotic cell processing equipment downstream of the device 100. FIG. 4 shows a schematic example of such a device 202. Thereby, exposure to contaminants is minimized and efficient aseptic processing of the amniotic fluid is possible in the post-filtration processing steps. FIG. 3 shows an example where the outlet connector 106 includes a tube 106 connected to the fluid outlet 104 at the first sealed connection portion 117. The outlet connector 106 can form a sealed connection with the fluid outlet 104 using a press-fit connection, an adhesive, a clamp, or other fixing elements. In another example, as schematically shown in FIG. 4, the outlet connector 106 is a continuous extension of the fluid outlet 104 without separate fixing elements, for example, formed as a single part by molding or other material forming techniques. FIGS. 3 and 4 show a second connector 118 configured to form a sealed connection with an amniotic cell processing device downstream of the device 100, such as a centrifuge 202. The second connector 118 may comprise a press-fit connection, a clamp, a combination thereof, or other releasable fixing elements. Thus, the connection between the second connector 118 and, for example, the centrifuge 202 can be repeatedly connected and disconnected and is resealable to maintain a sealed connection in such procedures. The chamber 102, the filter 101, the fluid inlet 103, the fluid outlet 104, the inlet connector 105, and the outlet connector 106 may be provided as a kit within a sterile package, for example, as a disposable kit. Such a kit, i.e., the device 100, provides an easy and improved process for filtering and processing amniotic stem cells in this way. The device 100 may comprise pumps 122, 123 arranged to pressurize the amniotic fluid to flow from the fluid inlet 103 to the fluid outlet 104. Thereby, more effective filtration of the amniotic fluid becomes possible. A larger volume can be filtered in a shorter time.

[0031] FIG. 6 shows an example in which a pump 122 is connected to a fluid outlet 104 so as to aspirate amniotic fluid in the direction of the arrow shown through a filter 101. The pump 122 may be arranged at a fluid inlet 103 so as to extrude amniotic fluid through the filter 101. The pump 122 can be a small manual pump integrated with the fluid inlet 103, the fluid outlet 104, the inlet connector 105, or the outlet connector 106.

[0032] FIG. 7 shows another example, to be described in more detail later, in which a pump 123 is arranged to pressurize amniotic fluid to flow from a fluid inlet 103 to a fluid outlet 104. The chamber 102 can include a conduit 119 disposed between the fluid inlet 103 and the fluid outlet 104. The pressure within the chamber 102 can be variable depending on the fluid and / or gas communication through the conduit 119. Thus, the flow of amniotic fluid through the filter 101 can be optimized according to the application. For example, the flow rate through the filter 101 can be increased or decreased by changing the pressure within the chamber 102 via the conduit 119.

[0033] Figure 5 shows an example where conduit 119 communicates with chamber 102. An access port 120, such as a connector or valve element, can be actuated to allow fluid or gas to be discharged from chamber 102 and / or injected into chamber 102, thereby affecting the pressure therein. Conduit 119 is disposed between fluid outlet 103 and filter 101 in Figure 5, but in another example, conduit 119 may be disposed between fluid inlet 103 and filter 101. Figure 5, described below, shows a further example of conduit 119 communicating with chamber 102. As illustrated in Figure 7, pump 123 can be disposed in communication with conduit 119. This facilitates optimization of the flow within chamber 102 and the associated filtration process. In the example of Figure 7, conduit 119 variably communicates with upstream cavity 108 of chamber 102 and downstream cavity 109 of chamber 102, i.e., filter 101 can be disposed to divide chamber 102 into upstream cavity 108 and downstream cavity 109. In Figure 7, conduit 119 is connected to both upstream cavity 108 and downstream cavity 109. Pump 123 is configured to pressurize amniotic fluid to flow from upstream cavity 108 to downstream cavity 109 or from downstream cavity 109 to upstream cavity 108. In the latter case, it can be advantageous in situations where, for example, an instantaneous reverse flow is desired to relieve clogging or blockage of filter 101. In such a case, as schematically shown in Figure 7, valves 120, 120', 121, 121' are operated to provide the desired flow direction. For example, in the case of reverse flow, valves 120 and 121' may be open and valves 120' and 121 may be closed. In normal filtration mode, valves 121, 121' may be open and valves 120, 120' may be closed. Upstream cavity 108 can also be pressurized by opening valve 120' in such a filtration mode.

[0034] As schematically shown in FIG. 8, the filter 101 may include a first filter element 101a and a second filter element 101b disposed between the first filter element 101a and the fluid outlet 104. The second filter element 101b may have a mesh diameter or pore system smaller than that of the first filter element 101a. Thereby, particulate matter with gradually decreasing dimensions can be effectively filtered. Thus, the risk of filter clogging is reduced. This enables a more reliable and robust filtration process for amniotic fluid. Improved filtration of amniotic fluid containing particulate matter in a wider size range is also provided. Furthermore, since stem cells are not lost in clogged pores, a larger fraction of stem cells in the amniotic fluid can be obtained. Although there are two filter elements 101a and 101b in FIG. 8, it should be understood that any plurality of filter elements with a gradually decreasing mesh diameter or pore system in the direction of fluid flow from the fluid inlet 103 to the fluid outlet 104 can be sequentially arranged in the chamber 102 for effective filtration of particulate matter with gradually decreasing dimensions. The first and second filter elements 101a and 101b may be separated by a distance (d) along the direction of the flow of amniotic fluid from the fluid inlet 103 to the fluid outlet 104, as schematically shown in the example of FIG. 8. The movement of the amniotic fluid between the first and second filter elements 101a and 101b may, in some cases, involve turbulent flow, which can further reduce the risk of undesirable accumulation of particles on the first and second filter elements 101a and 101b.

[0035] The filter 101 may include a mesh having a mesh diameter in the range of 20 to 2000 μm. In another example, the filter 101 includes a mesh having a mesh diameter in the range of 100 to 500 μm. This enables particularly effective filtration of particulate matter from the amniotic fluid. Returning again to FIG. 8, the first filter element 101a may include a mesh having a mesh diameter in the range of 500 to 1000 μm, and the second filter element 101b may include a mesh having a mesh diameter in the range of 30 to 150 μm. In this way, the first filter element 101a can remove larger debris, and subsequently, smaller particles are removed by the second filter element 101b. This further minimizes the risk of clogging, enabling particularly effective filtration of particulate matter of various sizes and a higher amount of reliable filtration over a longer period. As described above, any plurality of filter elements can be arranged continuously within the chamber 102.

[0036] FIG. 9 shows three filter elements 101a, 101b, 101c disposed within chamber 102. In some examples, filter elements having the smallest mesh diameter or pore system disposed furthest downstream of chamber 102, such as filter element 101b of FIG. 6 and filter element 101c of FIG. 9, may have a mesh diameter or pore system sized such that only a single amniotic cell or an amniotic cell mass smaller than 10 cells can pass through filter 101. The smallest mesh diameter or pore system in such an example can be about 30 μm. Filter 101 can include a mesh such as a nylon mesh. Filter 101 can include a porous material having a variable pore diameter that passes through filter 101 in the direction of the flow of amniotic fluid from fluid inlet 103 to fluid outlet 104. That is, the amniotic fluid flows in a direction through filter 101 toward outlet 104 and the pore diameter gradually decreases, so that larger debris is removed at the surface of filter 101 closest to inlet 103 while smaller sized particles are removed deeper within the filter. As described above, chamber 102 can include an upstream cavity 108 and a downstream cavity 109. The upstream and downstream cavities 108, 109 can be formed as integral parts for forming chamber 102, for example, in a molding process or by other material forming techniques. The upstream and downstream cavities 108, 109 can be formed as separate units and then connected to each other, for example, by an adhesive or welding, to form a sealed connection. Filter 101 can be attached simultaneously with or after such a welding process or by the aforementioned adhesive.

[0037] As schematically shown in FIG. 9, the upstream and downstream cavities 108, 109 can be releasably connected to each other by a connecting element 110 so as to form a sealed connection. Thereby, for example, in order to replace the filter 101, the chamber 102 can be opened. Thus, the filter 101 can be releasably connected to the chamber 102. For example, the filter elements 101a, 101b, 101c can be releasably connected to the chamber 102 of FIG. 7. Thereby, filter elements 101a, 101b, 101c having different pore diameters or mesh diameters, or different numbers of such filter elements, can be attached to the chamber 102, enabling easy customization for different applications.

[0038] The connection element 110 is configured to form a hermetic connection with the upstream and downstream cavities 108, 109 and can comprise an annular gasket extending around the peripheral portions of the upstream and downstream cavities 108, 109. The filter 101 can include cartridges of different numbers of filter elements 101a, 101b, 101c having different pore sizes that can be adjusted according to a particular amniotic fluid sample. For example, an assessment of the turbidity and cloudiness of the amniotic fluid (level of vernix caseosa in terms of both particle size and opacity) can be an indicator for using an appropriate filter cartridge. An attached chart for comparing amniotic fluid samples can indicate which filter cartridge to use. The upstream cavity 108 and / or the downstream cavity 109 can be funnel-shaped. FIGS. 3-9 show an example where both the upstream and downstream cavities 108, 109 are funnel-shaped. FIG. 11 shows an example where only the downstream cavity 109 is funnel-shaped. Having a funnel shape can be advantageous for guiding the flow of amniotic fluid along a desired symmetric vector through the filter 101 and the device 100. The upstream cavity 108 and / or the downstream cavity 109 can include chamber walls 111a, 111b arranged essentially parallel to the filter 101, i.e., perpendicular to the direction of flow of amniotic fluid from the fluid inlet 103 to the fluid outlet 104. FIG. 10 shows an example where the chamber walls 111a, 111b of the upstream and downstream cavities 108, 109 are arranged essentially parallel to the filter 101. This minimizes the space inside the chamber 102 while maintaining a sufficient filter area, for example, minimizing the risk of introducing air that can interfere with surfactant molecules, reducing the risk of infection, and decreasing the harmful formation of reactive oxygen species in amniotic cells. The chamber 102, and / or the inlet connector 105, and / or the outlet connector 106 can be formed from a phthalate ester-free PVC material. This provides a device suitable for contact with pharmaceutical starting materials such as amniotic cells.

[0039] The device 100 may comprise a protrusion 112 arranged to extend from the inner wall 113 of the chamber 102. FIGS. 11 and 12 show examples of such a protrusion 112 in a cross-sectional side view and through cross-section A-A, respectively. The protrusion 112 provides support to the filter 101 when the filter 101 begins to curve and bend towards the inner wall 113. Thus, since the filter 101 can be supported by the protrusion 112 away from the inner wall 113, flow through the mesh or pores of the filter 101 is still possible even in such cases. That is, the protrusion 112 can further suppress the risk of flow rate limitation and provide efficient, robust, and highly reliable filtration.

[0040] FIG. 13 is a flowchart of a method 300 for filtering amniotic fluid containing particulate matter and amniotic cells. The method 300 includes forming a sealed connection 301 between the fluid inlet 103 of the chamber 102 and the amniotic fluid sample source 201. The method 300 includes passing the amniotic fluid through the filter 101 enclosed in the chamber 102 by providing a flow of the amniotic fluid from the fluid inlet 103 to the fluid outlet 104 of the chamber 102. 302. Thereby, particulate matter is deposited on the filter 101, and the amniotic fluid containing amniotic cells flows through the outlet 104. Thus, the method 300 provides the advantageous benefits as described above in connection with the device 100 and FIGS. 3-12. The method 300 provides effective and aseptic filtration of amniotic fluid to obtain a high-quality amniotic cell sample.

[0041] In one embodiment, removing particulate matter from the TAF to obtain purified TAF cells can be performed by applying any method known in the art such as filtration, centrifugation, etc. The TAF can be filtered through a filter having a pore size of 20 μm or more. The filter can be made of any synthetic material including, but not limited to, cellulose acetate, cellulose nitrate (collodion), polyamide (nylon), polycarbonate, polypropylene, and polytetrafluoroethylene (Teflon). In one embodiment, the removal of particulate matter is performed by applying the device 100.

[0042] Next, select A variety of terms known to those of skill in the art are used throughout this specification and will continue to be used. For example, the terms "expressing, expression, and / or being expressed" in the context of cell surface markers indicate the presence of a particular marker on the surface of a cell and mean that the surface marker is being produced by the cell. Expression of a surface marker can be used to select between different cell populations. For example, positive selection for expression of a surface marker means selection of a cell population that expresses a particular surface marker more strongly compared to another cell population. Conversely, negative selection for expression of a cell surface marker means selection of a cell population that expresses a particular surface marker more weakly compared to another cell population.

[0043] As described above and elsewhere in this specification, TAFs include various progenitor cell types. In certain examples, a particular progenitor cell type can be isolated and expanded via adhesion selection. For example, Synthemax (Merck, CORNING®, Synthemax®, II - SC SUBSTRATE, CLS3535 - 1EA), a vitronectin substrate, can be used as a coating to create a more in vivo - like environment for stem cell culture, thereby restricting the maturation of TAF - derived progenitor cells and maintaining plasticity. Synthemax is a synthetic, animal - component - free, flexible vitronectin - based peptide substrate for serum - free or serum - containing growth of human progenitor cells / stem cells and other adult stem cell types. Those of skill in the art will understand that a vitronectin - based peptide substrate can include a portion of the vitronectin protein such as a specific peptide sequence of vitronectin. Alternatively, intact vitronectin protein may be used. The Synthemax vitronectin substrate provides a synthetic, xeno - free alternative to biological coatings and / or feeder cell layers commonly used in cell culture known in the art. Briefly, a standard tissue culture treated flask is coated with approximately 0.2 mL of Synthemax / cm 2Coated at (10 μg / mL) to obtain a surface density of 2 μg / cm² 2 and can be incubated at 37°C for about 1 hour, 0.5 hour, 2 hours, 4 hours, 8 hours, or more than 8 hours, or at room temperature for about 2 hours, 1 hour, 4 hours, 8 hours, or more than 8 hours, optionally with excess solution that is removed and replaced. In certain examples, Synthemax can be coated at a surface density of about 1 - 5 μg / cm² 2 , such as 2 μg / cm² 2 , 0.1 - 10 μg / cm² 2 , 0.5 - 4 μg / cm² 2 , 1 - 3 μg / cm² 2 , or about 1.5 - 2.5 μg / cm² 2 .

[0044] In other embodiments, the adhesion selection can be performed using, for example, a surface coated with collagen, fibronectin. Alternatively, the adhesion selection can be performed using an uncoated surface including tissue culture-treated plastic.

[0045] Cells purified from TAF solution can be gently resuspended in pre-warmed cell culture medium free of xenogeneic components, and then the cell suspension is added to a flask coated with Synthemax. The medium can be exchanged at various time points after addition to the flask, for example, at about 2 hours to 168 hours, 12 hours to 96 hours, 24 hours to 72 hours, 36 hours to 60 hours, 42 hours to 56 hours, or 48 hours later, and then can be exchanged about daily, every other day, every three days, every five days, once a week, once every two weeks, or less than once every two weeks. By repeatedly removing the used medium, non-adherent cells can be removed, thereby selecting MSCs by their adhesion affinity to the Synthemax-treated surface. The cells can be cultured for a certain period, for example, about 4 days, 7 days, 10 days, 11 days, 12 days, 13 days, 14 days, 18 days, 21 days, 28 days, or longer than 21 days. Optionally, in some examples, the cells can be cultured under hypoxic conditions, and hypoxic priming can change the metabolism of the proliferating cells, enhance the resistance to oxidative stress, thereby improving the possibility of engraftment, survival in the ischemic microenvironment, and angiogenesis of the transplanted MSCs. After culturing, the formed P0 colonies (colony-forming units - CFU) can be dissociated and pooled. After pooling, the remaining cells can mainly be non-tissue-specific MSCs. In certain examples, the pooled P0 cells can be gently resuspended in pre-warmed cell culture medium free of xenogeneic components and re-seeded into a tissue culture-treated flask without Synthemax for passage. The pooled cells can be seeded at a seeding density of about 100 - 10000 cells / cm 2 , 500 - 8000 cells / cm 2 , 1000 - 5000 cells / cm 2 , or about 2000 - 4000 cells / cm 2 . The medium can be exchanged about every 1 day, 2 days, 4 days, or 5 days or more. After a certain period such as about 2 days, 4 days, 7 days, or 8 days or more, the cells can be dissociated and recovered. As described below, further selective MSC isolation can be achieved.

[0046] Identification of Biomarkers When comparing the gene expression profiles of TAF-MSCs and adult MSCs derived from adipose tissue or bone marrow by RNAseq, TAF-MSCs tend to express more of some genes present in adult MSCs and less of other genes. Identification of both positive and negative TAF-MSC-specific neonatal cell surface markers enables the selection of neonatal-quality MSCs from more differentiated cells of lower importance as progenitor cells using ligands such as antibodies and aptamers or other selection techniques.

[0047] Cell surface markers that distinguish tissue-related cells from other MSCs can be elucidated through a bioinformatics process utilizing a tissue-specific score algorithm. An example of the MSC tissue-specific score algorithm is shown in FIG. 14. Tissue specificity can be measured as a combination of two elements: "tissue transcriptional similarity", also known as the similarity score, and "tissue-specific gene expression program", also known as the gene set score. In a particular example, the similarity score can be the average Spearman correlation to a reference sample of each MSC tissue (e.g., a fetal lung MSC sample). In the example, the gene set score can be the average expression of genes in a tissue-specific gene set. As shown in FIG. 14, in a particular example, after normalizing the similarity score and the gene set score using the Z-transform to convert the input values, which are real or complex numbers, into a complex frequency domain representation, combining them, assigning equal weights to each score, and converting the combined value using the Z-transform, the resulting output is the MSC tissue-specific score. The MSC tissue-specific score evaluates the relative tissue specificity among input samples by measuring how many standard deviations of samples that are virtually specific to a given tissue there are compared to the average input sample. For example, the MSC tissue-specific score can indicate how much a clone sample appears to have a tissue-specific phenotype such as a lung phenotype compared to an average clone. Such an approach makes it possible to identify the top X% percentile score using a normal distribution function, which is in fact the top X% of clones that are most tissue-specific to the relevant tissue.

[0048] In one example, for a given tissue, the tissue-preferred clone can be defined as any clone belonging to the top X% percentile score, where X is any percentage within a range having a lower limit of about 0.1 to 25, such as about 1, 5, 10, 15, and 20, and an upper limit of about 30 to 75, such as about 35, 40, 45, 50, 55, 60, 65, or 70. An example of the results of the prioritization of TAF-MSC tissue specificity is shown in FIG. 15, where the 15% and 5% thresholds are evident. The tissue-specific clones can be prioritized, and then candidate surface marker genes can be identified. For each tissue, two groups can be defined: a tissue-preferred group and a tissue-low-preferred group. This determination can be made using a suitable analysis program, such as DEseq2 from Bioconductor.org. The tissue-preferred group can include clones with scores in the top 15% percentile. The tissue-low-preferred group can include clones in the lower Y% percentile, where Y is any percentage within a range having a lower limit of about 25 to 70, such as about 3, 35, 40, 45, 50, 55, 60, or 65, and an upper limit of about 75 to 99.9, such as about 80, 85, 90, 95, or 99. FIG. 16 shows an example of such an analysis for kidney tissue. Next, genes differentially expressed between the tissue-preferred group and the tissue-low-preferred group can be identified. Finally, the results of differential expression can be annotated with surface marker gene information.

[0049] In certain examples, to identify tissue-specific cell surface markers, the Z-fold increase in expression of preferential clones compared to average clones (Z is at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 8-fold, 10-fold, 12-fold, 15-fold, or even greater fold increase in expression) (log2FoldChange), and surface marker genes having Transcripts Per Kilobase Million (TPM) greater than about 500, such as greater than about 1000, 1500, 2000, 2500, 3000, 5000, or even greater, are selected to obtain top tissue-specific marker candidates, such as approximately the top 5, 10, 20, 30, 40, 50, 60, 70, 100 or more, as shown in Tables 3-6 below and described in more detail later. Suitable log2FoldChange and TPM values can further vary depending on the presence or absence of good markers and tissue type specificity.

[0050] After adhesion selection and passage, applying the above tissue-specific algorithm for identifying surface markers, TAF-MSC cells can express various identified surface markers as shown in Table 1 below, which suggests non-tissue-specific TAF MSC. Those skilled in the art will understand that such surface markers can be present at various surface densities and can be upregulated or downregulated compared to other cell types. Thus, such surface markers can be used to identify and isolate specific cell types. In some cases, the surface markers listed in Table 1 below may be expressed on average at least 8-fold higher in TAF MSC compared to other MSC cell types, particularly adult MSC derived from bone marrow or adipose tissue. The thresholds used to generate Table 1 are as follows: X was selected as 15%, Y was selected as 50%, Z was selected as 8-fold, and TPM greater than 3000 was selected. Those skilled in the art will understand that the numbering used in Table 1 and all tables in this specification is only used to indicate the total number of identified markers and does not indicate that a particular marker is more strongly expressed and / or preferred compared to another marker.

Table 1-1

Table 1-2

[0051] As will be understood by those skilled in the art, suitable combinations of the markers listed in Table 1 can be used to isolate TAF-MSCs from adult MSCs by selecting a specific marker from Table 1 or a combination of two, three, four, five, six, or more markers from Table 1. In certain examples, TAF MSCs can be more specifically identified by identifying a combination of stronger expression, such as a combination of stronger expression by 8-fold or more, of the aforementioned markers, such as TBC1D3K and / or AIF1L and / or CDHR1 and / or NKAIN4 and / or ABCB1 and / or PLVAP, compared to adult MSCs. When using a combination of markers, the identification can be achieved at a lower threshold of stronger expression, such as 2-fold or more, 4-fold or more, or 6-fold or more expression of each marker.

[0052] In contrast to the above surface markers (positive markers) that can be more strongly expressed on the surface of TAF-MSCs compared to adult MSCs, in certain examples, the following surface markers (negative markers) in Table 2 are expressed more weakly on TAF-MSCs compared to other cell types, such as expression of 1 / 8-fold or less of any combination of the aforementioned markers relative to adult MSCs (optionally with a TPM threshold > 500): IL13RA2, CLU, TMEM119, CEMIP, and LSP1. When using a combination of negative markers, the identification can be achieved at a lower threshold of weaker expression, such as 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less expression of each marker.

[0053] Combinations of two or more of these negative markers can also be used to more specifically isolate TAF MSCs. Furthermore, one of ordinary skill in the art will also recognize that combinations comprising both negative and positive markers, such as any of the above thresholds, may also be effective to more specifically isolate TAF MSCs. [Table 2]

[0054] Marker-based selection Amniotic fluid contains heterogeneous cells in a homogeneous liquid. Therefore, marker-based selection may be required. An example of marker-based selection is by the use of fluorescence-activated cell sorting (FACS). Fluorescence-activated cell sorting (FACS) can be used to purify the cell population of TAF-MSCs, and even if the target cell type expresses very low levels of identification markers and / or separation is required based on differences in marker density, FACS enables a very high purity of the desired cell population. FACS enables the purification of individual cells based on size, granularity, and fluorescence. As will be understood by those skilled in the art, FACS can be used to select a specific cell population that expresses more of a certain cell surface marker than another cell population, and vice versa. In some examples of purification methods, bulk purification methods such as panning, complement depletion, and magnetic bead separation can be used in combination with FACS or as an alternative to FACS. Briefly, to purify the cells of interest by FACS, first, they are stained with fluorescently tagged monoclonal antibodies (mAbs) that recognize specific surface markers on the desired cell population. Negative selection of unstained cells can also enable separation. In the case of GMP production of cells according to some examples, FACS can be performed using sorting technologies of closed systems such as MACSQuant® Tyto®. The sample can be kept uncontaminated within a disposable, fully enclosed MACSQuant Tyto cartridge. Furthermore, filtered air can send cells into the microchip through the microchannel at a very low pressure (<3 PSI). However, before entering the microchannel, potential cell aggregates can be suppressed by a filter system, and a fluorescence detection system that guarantees a smooth sorting process can detect the cells of interest based on the predetermined fluorescence parameters of the cells. Based on the fluorescence and scattered light characteristics of the target cells, their directions can be changed by a sorting valve located within the microchannel. In a specific example of the purification method, the success of staining and thus sorting can depend greatly on the selection of identification markers and the selection of mAbs.The sorting parameters can be adjusted according to the requirements of purity and yield. Different from conventional droplet sorters, the cells sorted by MACSQuant Tyto do not have to experience high pressure or charging, nor do they have to be depressurized. Thus, such a gentle sorting approach can result in high cell viability and functionality. Alternatively, other marker-based selection techniques may be known to those skilled in the art. These include, but are not limited to, magnetic-activated cell sorting, microfluidics-based sorting, buoyancy-activated cell sorting, mass cytometry, and the like.

[0055] Tissue-specific cells and usage Lung TAF cell markers As described above, RNAseq data from TAF-MSC clones, adult and neonatal MSC reference materials, and fetal fibroblasts, along with analysis of publicly available expression datasets, can be used to identify and characterize TAF-MSC cells. For example, subpopulations of TAF-MSCs can be established by clustering their expression data (RNAseq) with neonatal reference samples. Such subpopulations include, but are not limited to, lung MSCs, urinary tract MSCs (also described as kidney MSCs in this disclosure), and skin MSCs. Gene lists of highly and lowly expressed genes for each cluster of expression data may enable identification of surface marker genes for each cluster. Using such data comparisons, subpopulations of TAF cells were compared to adult MSC cells based on their gene expression (RNAseq), and a list of neonatal-specific surface marker genes for each cluster was obtained. Several surface markers of interest associated with lung TAF cells were identified. For example, a non-exclusive list of preferred surface markers used to identify and isolate lung TAF cells is provided below. Furthermore, since the number of different MSC subtypes of TAF is limited, selection of tissue-specific MSCs can be done by first characterizing and then stepwise negatively selecting / classifying the material considering the (multivariate) surface marker profiles of combinations of different tissue-specific MSCs. One of ordinary skill in the art will understand that any such combination of these surface markers can be used for the identification and isolation of lung TAF cells from a general population of TAF-derived cells and / or TAF-MSC cells. In some examples, the surface markers of the following non-exclusive list may be more highly expressed on the surface of lung TAF cells compared to other cell types such as other TAF-derived cells and / or other TAF-MSC cells.

[0056] As described above, bioinformatics techniques can be used to identify tissue-specific surface markers, and thus the surface markers identified in Table 3 may have at least a 10-fold increase in expression in preferred clones compared to average TAF-MSC clones (optionally with a TPM threshold > 2000).

Table 3

[0057] In contrast to the above surface markers that may be more strongly expressed on the surface of lung TAF MSCs, in certain examples, the following surface markers may be less strongly expressed in lung TAF MSCs compared to other TAF-derived cells and / or other cell types such as TAF-MSCs: CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR. As will be understood by those skilled in the art, one, two, three, four, or more of the aforementioned less strongly expressed surface markers can be used to isolate lung TAF cells from other TAF-derived cells and / or other cell types such as TAF-MSCs.

[0058] In certain examples, the cell surface marker CD248 (endosialin) can be used to sort lung TAF MSCs from a population of TAF MSCs. Additional surface markers that can be used for the sorting of lung TAF MSCs include DDR-1 (discoidin domain receptor tyrosine kinase 1) and LRRC38 (leucine-rich repeat-containing protein 38), and all three of these have been identified by antibodies as markers useful for separation. In some examples, endosialin, DDR-1, and / or LRRC38 can be used alone or in combination with other markers for sorting. Endosialin can be sorted in combination with DDR-1 or LRRC38, or DDR-1 and LRRC38 can be combined without endosialin.

[0059] As will be understood by those skilled in the art, by selecting specific markers from Table 3, or combinations of two, three, four, five, six, or more markers from Table 3 and / or CD248 and / or DDR-1 and / or LRR38, suitable combinations of the markers listed in Table 3 with CD248, DDR-1, and LRR38 can be used to isolate lung TAF MSCs from TAF MSCs. In certain examples, lung TAF MSCs can be more specifically identified by identifying a combination of stronger expression, such as more than 10-fold stronger expression (optionally with a TPM threshold > 2000), of any combination of the aforementioned markers, such as PCDH19 and / or DDR1 and / or MME and / or IFITM10 and / or BGN and / or NOTCH3 and / or CD248 and / or DDR-1 and / or LRR38, compared to TAF MSCs. When using combinations of markers, the identification can be achieved at a lower threshold of stronger expression, such as more than 4-fold, more than 6-fold, or more than 8-fold expression of each of the markers.

[0060] In contrast to the above surface markers (positive markers) that can be more strongly expressed on the surface of lung TAF MSCs compared to TAF MSCs, in certain examples, the following surface markers (negative markers) are expressed more weakly on lung TAF MSCs compared to other cell types, such as expression that is 1 / 8-fold or less (optionally with TPM > 500) of any combination of the aforementioned markers relative to TAF MSCs: CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR. When using combinations of negative markers, the identification can be achieved at a lower threshold of weaker expression, such as expression that is 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less of each of the markers.

[0061] Combinations of two or more of these negative markers can also be used to more specifically isolate lung TAF MSCs. Furthermore, one of ordinary skill in the art will also recognize that combinations containing both negative and positive markers, such as any of the above thresholds, may also be effective in more specifically isolating lung TAF MSCs.

[0062] Figures 17A - 17D show an example of the results of a proof - of - concept study regarding the potential use of lung TAF MSCs for therapy, performed using neonatal - selected TAF MSCs (designated "LBX - THX - 001") that express MSC cup cell - surface markers including CD248, DDR1, and LRRC38. The purpose of this study was to investigate the effect of LBX - THX - 001 cells in a bleomycin - induced pulmonary fibrosis model in male rats. Two cell concentrations (2M cells / kg and 5M cells / kg) and two vehicles for the cells (PBS and CryoStor CS - 10) were tested.

[0063] The development of fibrosis in rat lungs after exposure to bleomycin is well - documented in the literature and is a model frequently used to study the effects of different treatments along with the pathology of pulmonary fibrosis. The number of LBX - THX - 001 cells injected was chosen to be appropriate for possible human therapy. Thus, the number of cells was chosen to reflect the number of cells used in previous studies regarding rats (8 - 20M cells / kg) and humans (0.5 - 2M cells / kg).

[0064] Pulmonary fibrosis was induced in 34 male SD rats by intratracheal injection of bleomycin (1000 U / rat). During the first week, the rats were monitored daily for body weight measurement, and thereafter, body weight was measured twice a week until the end of the test. Four days after bleomycin challenge, LBX-THX-001 cells were administered by intravenous (iv) injection. The injection volume was 194 - 535 μL (maximum allowable injection volume 1 mL / kg). The reaction to intratracheal injection of bleomycin was as expected based on previous experience with a model in which body weight decreased during the first few days after injection and then recovered. No significant difference in body weight loss was observed between the bleomycin group and the treatment group.

[0065] As shown in FIGS. 17A - D, the intravenous infusion of bleomycin induced fibrotic changes in the lung. Histopathological evaluation concluded the pathological changes in the bleomycin group both in terms of the proportion of affected parenchyma and after scoring using the modified Ashcroft scale. As shown in FIGS. 17A - D, the group treated with LBX-THX-001 cells (2 million cells / kg) 4 days after bleomycin showed significantly less pulmonary fibrosis compared to the bleomycin group. This was seen in both histopathological evaluation using the reading of "proportion of affected parenchyma" (FIGS. 17A - B) and the Ashcroft modified scale for fibrosis scoring (FIGS. 17A - D). Human MSCs could not be detected in the lungs of the rats at the end (28 days later).

[0066] Renal TAF cell marker Similar to the lung TAF MSC cell markers identified above, several surface markers for several purposes related to kidney TAF cells were identified. For example, a non-exclusive list of surface markers used to identify and isolate kidney TAF MSCs is shown in Table 4 below. Similar to the lung TAF MSC markers, the surface markers identified in Table 4 can have at least a 12-fold increase in expression in preferential kidney TAF clones compared to average TAF-MSC clones (optionally with a TPM threshold > 2000). Furthermore, since the number of different MSC subtypes of TAF is limited, the selection of tissue-specific MSCs can be done by first characterizing and then stepwise negatively selecting / classifying the material considering the (multivariate) surface marker profiles of combinations of different tissue-specific MSCs. Those skilled in the art will understand that any such combination of these surface markers can be used for the identification and isolation of kidney TAF cells from a general population of TAF-derived cells and / or TAF-MSC cells. In some examples, the surface markers in the following non-exclusive list can be more highly expressed on the surface of kidney TAF cells compared to other cell types such as other TAF-derived cells and / or other TAF-MSC cells. [Table 4]

[0067] As will be understood by those skilled in the art, by selecting specific markers from Table 4, or combinations of 2, 3, 4, 5, 6, or more markers from Table 4, suitable combinations of the markers listed in Table 4 can be used to isolate kidney TAF cells from TAF-MSCs. In certain examples, kidney TAF MSCs can be more specifically identified by identifying a combination of stronger expression of the aforementioned markers, such as any combination of HAVCR1 and / or CD24 and / or CLDN6 and / or ABCB1 and / or SHISA9 and / or CRB3, compared to TAF MSCs, for example, a combination of stronger expression by 12-fold or more (optionally with a TPM threshold > 2000). When using combinations of markers, identification can be achieved at lower thresholds of stronger expression, such as 4-fold or more, 6-fold or more, or 8-fold or more expression of each marker.

[0068] In contrast to the above surface markers (positive markers) that can be more strongly expressed on the surface of kidney TAF MSCs, in certain examples, the following surface markers (negative markers) are more weakly expressed in kidney TAF cells compared to other cell types, for example, expression that is 1 / 8-fold or less of any combination of the aforementioned markers relative to other TAF-derived cells and / or TAF-MSC cells (optionally with a TPM threshold > 500): GREM1, PDGFRB, BGN, FAP, CXCL12, CCKAR, CD248. When using combinations of negative markers, identification can be achieved at lower thresholds of weaker expression, such as 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less expression of each marker.

[0069] Combinations of two or more of these negative markers can also be used to more specifically isolate kidney TAF MSCs. Furthermore, those skilled in the art will also recognize that combinations containing both negative and positive markers, such as at any of the above thresholds, can also be effective in more specifically isolating kidney TAF MSCs.

[0070] Skin TAF cell markers Similar to the lung and kidney TAF MSC markers identified above, several surface markers for several purposes related to skin TAF cells were identified. For example, a non-exclusive list of surface markers used to identify and isolate skin TAF cells is shown in Table 5 below. The skin TAF MSC markers identified in Table 5 can have at least a 12-fold increase in expression in preferred clones compared to average TAF-MSC clones (optionally with a TPM threshold > 2000). Furthermore, since the number of different MSC subtypes of TAF is limited, the selection of tissue-specific MSCs can be done by first subjecting the material to stepwise negative selection / classification based on characterization and then considering the (multivariate) surface marker profiles of combinations of different tissue-specific MSCs. Those skilled in the art will understand that any such combination of these surface markers can be used for the identification and isolation of skin TAF cells from a general population of TAF-derived cells and / or TAF-MSC cells. In some examples, the surface markers in the following non-exclusive list can be more highly expressed on the surface of skin TAF cells compared to other cell types such as other TAF-derived cells and / or other TAF-MSC cells.

Table 5

[0071] As will be understood by those skilled in the art, suitable combinations of the markers listed in Table 5 can be used to isolate skin TAF-MSCs from TAF-MSCs by selecting a specific marker from Table 5 or a combination of two, three, four, five, six, or more markers from Table 5. In certain examples, skin TAF MSCs can be more specifically identified by identifying a combination of stronger expression of the aforementioned markers, such as any combination of TNFSF18 and / or PCDH19 and / or NCAM2 and / or TNFSF4 and / or CD248 and / or DDR2, for example, stronger expression by 12-fold or more (optionally TPM > 2000), compared to TAF-MSCs. When using a combination of markers, the identification can be achieved at a lower threshold of stronger expression, such as 4-fold or more, 6-fold or more, or 8-fold or more expression of each marker.

[0072] In contrast to the above surface markers (positive markers) that can be more strongly expressed on the surface of skin TAF cells, in certain examples, the following surface markers (negative markers) are expressed more weakly on skin TAF cells compared to other cell types, for example, expression of 1 / 8-fold or less of any combination of the aforementioned markers relative to other TAF-derived cells and / or TAF-MSC cells (optionally TPM threshold > 500): CD24, TNFSF10, ITGB4, ABCB1. When using a combination of negative markers, the identification can be achieved at a lower threshold of weaker expression, such as 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less expression of each marker.

[0073] Combinations of two or more of these negative markers can also be used to more specifically isolate skin TAF MSCs. Furthermore, those skilled in the art will also recognize that combinations containing both negative and positive markers, at any of the above thresholds, for example, can also be effective in more specifically isolating skin TAF MSCs.

[0074] Neural TAF cell marker Similar to the lung, kidney, and skin TAF MSC markers identified above, several surface markers of interest associated with neural TAF cells were identified. For example, a non-exclusive list of surface markers used to identify and isolate neural TAF cells is shown below. Neural TAF MSC surface markers identified in Table 6 can have at least a three-fold increase in expression in preferential clones compared to average TAF-MSC clones (optionally with a TPM threshold > 500). Furthermore, since the number of different MSC subtypes of TAF is limited, the selection of tissue-specific MSCs can be done by first characterizing and then stepwise negatively selecting / classifying the material considering the (multivariate) surface marker profiles of combinations of different tissue-specific MSCs. One skilled in the art will understand that any such combination of these surface markers can be used for the identification and isolation of neural TAF cells from a general population of TAF-derived cells and / or TAF-MSC cells. In some examples, the surface markers of the following non-exclusive list can be more highly expressed on the surface of neural TAF cells compared to other cell types such as other TAF-derived cells and / or TAF-MSC cells.

Table 6

[0075] As will be understood by those skilled in the art, suitable combinations of the markers listed in Table 6 can be used to isolate neural TAF-MSCs from TAF-MSCs by selecting a specific marker from Table 6 or a combination of two, three, four, five, six, or more markers from Table 6. In certain examples, neural TAF MSCs can be more specifically identified by identifying a combination of stronger expression of any of the aforementioned markers, such as HAVCR1 and / or ACKR3 and / or OSCAR and / or C3 and / or SIRPB1 and / or SLC6A6, compared to TAF-MSCs, for example, stronger expression by more than 3-fold (optionally with a TPM threshold > 500). When using a combination of markers, the identification can be achieved with a lower threshold of stronger expression, such as more than 2-fold of each marker, or a higher threshold of expression such as more than 6-fold, 8-fold, or 12-fold. Furthermore, those skilled in the art will also recognize that combinations containing both negative and positive markers, at any of the above thresholds, can also be effective in more specifically isolating neural TAF MSCs.

[0076] All features disclosed herein (including any attached sheets, claims, abstract, and drawings), and / or all steps of any method or process so disclosed, can be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The present disclosure is not limited to the details of any of the foregoing examples. The present disclosure extends to any novel one or any novel combination of the features disclosed herein (including the accompanying claims, abstract, and drawings), or to any novel one or any novel combination of the steps of any method or process so disclosed.

[0077] One of ordinary skill in the art will understand that, in some instances, the actual steps performed in the illustrated or disclosed process may differ from those shown in the figures. Depending on the example, some of the above steps may be deleted, while others may be added. For example, the actual steps taken or the order of steps in the disclosed process may differ from those shown in the figures. Depending on the example, some of the above steps may be deleted, while others may be added. Further, the features and attributes of the specific embodiments disclosed above can be combined in different ways to form additional embodiments, all of which are included within the scope of the present disclosure.

[0078] Conditional language such as "can", "could", "might", or "may", unless otherwise specified or understood differently within the context in which it is used, generally conveys that a particular instance includes a particular function, element, or step, while other instances do not. Thus, such conditional language generally does not imply that a function, element, or step is necessarily required in one or more instances, or that one or more instances necessarily include logic for determining whether these functions, elements, or steps are included in or performed by any particular instance, regardless of the presence or absence of user input or prompts. Terms such as "comprising", "including", "having", etc. are synonyms and are used inclusively in an unrestricted manner, excluding no additional elements, functions, acts, operations, etc. Also, the term "or" is used in its inclusive sense (not in its exclusive sense), so that, for example, when used to connect a list of elements, the term "or" means one, some, or all of the elements in the list. Similarly, the term "and / or" in relation to a list of two or more items encompasses all of the following interpretations: any one of the items in the list, all of the items in the list, and any combination of the items in the list. Further, the term "each" as used herein, in addition to having its ordinary meaning, can mean any subset of the set of elements to which the term "each" applies. Additionally, the words "herein", "above", "below", and words of similar import, when used herein, refer to the specification as a whole, not to any particular part of the specification.

[0079] Connective language such as the phrase "at least one of X, Y, and Z" is understood in the context in which it is generally used to convey that an item, term, etc. can be any of X, Y, or Z, unless otherwise specified. Thus, such connective language generally does not purport to imply that a particular instance requires the presence of at least one of X, at least one of Y, and at least one of Z.

[0080] As used herein, language such as the terms "substantially", "about", "generally", and "essentially" is used to denote a value, quantity, or property that is close to the value, quantity, or property described and still performs the desired function or achieves the desired result. For example, the terms "substantially", "about", "generally", and "essentially" may refer to an amount within less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the amount described. As another example, in certain instances, the terms "substantially parallel" and "essentially parallel" refer to a value, quantity, or property that deviates from exact parallel by 15 degrees, 10 degrees, 5 degrees, 3 degrees, 1 degree, or 0.1 degree or less.

[0081] Various modifications to the embodiments described in this disclosure may be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments without departing from the spirit or scope of this disclosure. Thus, this disclosure is not intended to be limited to the embodiments shown herein, but rather should be accorded the widest scope consistent with the principles and features disclosed herein. Specific examples of this disclosure are listed below or are included in a set of claims to be presented in the future.

Claims

1. A method for obtaining term amniotic fluid cells (TAF cells) from term amniotic fluid, comprising: providing term amniotic fluid (TAF); removing particulate matter from the TAF to obtain purified TAF cells; performing adhesion selection of the purified TAF cells to obtain TAF adherent cells; subculturing the TAF adherent cells to obtain a population of cells containing the TAF cells; selecting TAF cells that further express DDR1 and LRRC38 from the population as cells expressing a surface marker that is at least CD248; and wherein the TAF cells are mesenchymal stem cells derived from term amniotic fluid.

2. The method according to claim 1, further comprising selecting the TAF cells from the population as cells expressing at least one marker selected from the group consisting of PCDH19, MME, IFITM10, BGN, NOTCH3, SULF1, TNFRSF18, BDKRB1, FLT1, PDGFRA, TNFRSF4, UNC5B, FAP, CASP1, DDR2, PCDH18, and CRLF1.

3. The method according to claim 1 or 2, wherein selecting the TAF cells comprises excluding cells expressing a marker selected from the group consisting of CD24, ITGB4, TNFRSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR.

4. The method according to any one of claims 1 to 3, wherein removing particulate matter comprises filtering and centrifuging the TAF.

5. The method according to any one of claims 1 to 4, wherein performing adhesion selection of the purified TAF cells comprises adhering the purified TAF cells to a surface coated with a vitronectin-based substrate.

6. The method according to any one of claims 1 to 5, wherein the selection step is performed using fluorescence-activated cell sorting (FACS).

7. The method according to any one of claims 1 to 6, wherein the selection step is performed using an antibody against any of the markers or surface markers.

8. The method according to any one of claims 1 to 7, wherein the selection step comprises selecting TAF cells that express at least two markers, at least three markers, or at least four markers from the surface markers according to claim 2.

9. The method according to any one of claims 1 to 8, wherein the selection step comprises a plurality of sorting steps, and each sorting step assigns TAF cells to a first output group or a second output group according to a set of markers expressed or not expressed by each TAF cell.

10. The selection step is a first sorting step for assigning TAF cells expressing a surface marker that is at least CD248 to a first output group; and a second sorting step for assigning TAF cells from the first output group expressing a second set of markers to a second output group, the method according to claim 9.

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